Epitopes specificity of antibodies to thyroid peroxidase in patients with Graves' disease, Hashimoto's thyroiditis and overlap-syndrome.

Background: Antibodies against thyroid peroxidase (anti-TPO) serve as clinical markers of thyroid autoimmune diseases (TAIDs). By trying to elucidate the causes of heterogeneity in autoantibody levels among patients with different TAIDs it becomes possible to clarify the pathophysiology of GD and HT. Objective: To investigate the heterogeneity of epitopes recognized by anti-TPO in patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and overlap-syndrome. Methods: We carried out a cross-sectional study on 398 patients with GD, HT and overlap syndrome and analyzed the specificity of epitopes and binding constants of TPO with monoclonal antibodies (MAbs). Ten MAbs to TPO were used, of which five were reactive with native TPO and the rest were reactive with denaturated TPO. Results: The autoantibodies in blood serum of HT patients inhibited the binding of MAb63 more significantly than those in serum of GD patients: 59.62 % versus 54.02 %, respectively (p = 0.001). The anti-TPOs in serum of GD patients inhibited the binding of MAb77 more significantly than those in serum of HT patients: 54.36 % versus 51.13 %, respectively (p = 0.047). The binding of MAb45 was more inhibited in serum of patients with anti-TPO concentration over 1000 IU/ml (58.36 %). The blood serum of patients with overlap-syndrome showed less significant inhibition of MAb63 binding than that of patients with no overlap-syndrome: 52.47 % versus 58.81 %, respectively (p = 0.043). Conclusion: Mapping the epitopes to TPO with the help of MAbs may improve the differential diagnosis between different thyroid autoimmunities.

Gene hypermethylation in blood leukocytes in humans long term after radiation exposure - Validation set.

Hypermethylation of СpG islands in the promoter regions of several genes with basic protective function in blood leukocytes of individuals exposed to ionizing radiation long time ago (2-46 years), and differential effects of age and radiation exposure on hypermethylation was reported in our previous work. To validate these results, epigenetic modifications were assessed in an independent series of 49 nuclear industry workers from the "Mayak" facility (67-84 years old at sampling) with documented individual accumulated doses from the prolonged external γ-radiation exposure (95.9-409.5 cGy, end of work with radiation:0.3-39 years ago), and in 50 non-exposed persons matched by age. In addition to the genes analyzed before (RASSF1A, p16/INK4A, p14/ARF, GSTP1), four additional loci were analyzed: TP53, ATM, SOD3, ESR1. The frequency of individuals displaying promoter methylation of at least one of the 8 genes (71.4%) was significantly higher in exposed group as compared to the control group (40%), p = .002, OR = 3.75. A significantly elevated frequency of individuals with hypermethylated СpG islands in GSTP1, TP53, SOD3 promoters was revealed among exposed subjects as compared to the control group (p = .012, OR = 8.41; p = .041, OR = 4.02 and p = .009, OR = 3.42, respectively). A similar trend (p = .12, OR = 3.06) was observed for the p16/INK4A gene. As a whole, p16/INK4A and GSTP1 promoter hypermethylation in irradiated subjects from both previously and currently analyzed groups was pronounced. Thus, the direction of the effects was fully confirmed, suggesting the result reproducibility. No statistically significant correlation between promoter methylation and individual radiation dose was found. Further studies are required to create an array of blood epigenetic markers of radiation exposure associating with premature aging and age-related diseases and to accurately evaluate radiation-added effect across the range of doses. SYNTHESIS: The results of studies of epigenetic changes in two independent samples of irradiated subjects indicated the significance of radiation factor in the induction of hypermethylation of CpG islands in gene promoters that is revealed in blood cells years and decades after exposure.

Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (ß=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (ß=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development of age-associated cancer and non-cancer diseases.

Analysis of genomic instability in the offspring of fathers exposed to low doses of ionizing radiation.

Transgenerational genomic instability was studied in nonirradiated children born from fathers who were irradiated with low doses of ionizing radiation while working as clean-up workers at the Chernobyl Nuclear Power Plant (liquidators) and nonirradiated mothers from nuclear families. Aberrant cell frequencies (ACFs), chromosomal type aberration frequencies, and chromatid break frequencies (CBFs) in the lymphocytes of fathers-liquidators, and their children were significantly higher when compared with the control group (P < 0.05). Individual ACFs, aberration frequencies, and CBFs were independent of the time between irradiation of the father and conception of the child (1 month to 18 years). Chromosomes were categorized into seven groups (A through G). Analysis of aberrant chromosomes within these groups showed no differences in the average frequency of aberrant chromosomes between children and fathers-liquidators. However, significant differences were observed in the average frequency of aberrant chromosomes in groups A, B, and C between children and mothers in the families of liquidators. These results suggest that low doses of radiation induce genomic instability in fathers. Moreover, low radiation doses might be responsible for individual peculiarities in transgenerational genomic instability in children (as a consequence of response to primary DNA damage). Thus, genomic instability may contribute to increased morbidity over the lifetime of these children.

Development of ultra-sensitive soybean peroxidase-based CL-ELISA for the determination of human thyroglobulin.

Serum thyroglobulin (Tg) is a main marker of thyroid cancer relapses after total or near-total thyroidectomy of patients with differentiated thyroid carcinoma. In this study, we developed a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting Tg in human serum. Soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) and horseradish peroxidase (HRP) with p-iodophenol (PIP) were used as detection systems in the sandwich CL-ELISA. Comparison of these two systems showed that a lower detection limit (LOD) of CL-ELISA with SbP/SPTZ/MORPH was 10 times lower than for the immunoassay with HRP/PIP. The LOD value for SbP-based CL-ELISA of 0.2 ng/mL was identical to LOD value typical of CL-ELISA Immulite kit produced with alkaline phosphatase. The sensitivity of Tg CL-ELISA using SbP/SPTZ/MORPH completely satisfies the requirements of modern endocrinology. Comparative study of clinical serum specimens assayed by the SbP-based CL-ELISA (x) and Immulite kit (y) for detecting Tg showed a good correlation between these two immunoassays (y=1.15 x -0.14, R=0.99). The obtained results open good perspectives for use of SbP/SPTZ/MORPH system in the development of ultra-sensitive immunoassays.

Fumonisins B1 and B2 in agricultural products consumed in South Korea: an exposure assessment.

To survey fumonisins B1 (FB1) and B2 (FB2) in agricultural products consumed in South Korea and provide an exposure assessment, ground samples were extracted (80% MeOH), filtered (0.2 microm), and cleaned up. After evaporation, dry residues were reconstituted in 50% MeOH, and a 50-micro1 aliquot of this sample was mixed with 200 micro1 of o-phthaldialdehyde for derivatization. The derivatives were analyzed with a high-performance liquid chromatography system equipped with a fluorescence detector. For validation of the detection procedure, linearity, accuracy, precision, detection limit, and quantification limit were determined. The validated detection method was then used to survey fumonisins in white rice, brown rice, barley, barley tea, beer, wheat flour, millet, dried corn, corn flour, corn tea, canned corn, popcorn, and breakfast cereal. Retention times for FB1 and FB2 standards were 7 and 18 min, respectively. Linearity (R2 = 0.99995 to 0.99998), accuracy (81.47 to 108.83%), precision (2.35 to 5.77), detection limit (25 ng/g or ng/ml), and quantification limit (37 ng/g or ng/ml) indicated that this procedure is capable of quantifying fumonisins in agricultural products. Only FB1-positive samples (5.12%, three dried corn samples and five corn flour samples) were found at 90.89 to 439.67 ng/g. According the survey results, an estimated daily intake of FB1 and FB2 in Korea was 0.087 ng/kg of body weight per day. These results indicate that continuous monitoring of these mycotoxins is necessary to establish appropriate risk assessment, and the maximum tolerable daily intake of fumonisins in Korea is lower than the 2 microg/kg set by the Joint Food and Agriculture Organization-World Health Organization Expert Committee.